Leptospirosis is a global, zoonotic disease caused by members of the genus Leptospira. Although widespread and sometimes fatal, leptospirosis is considered a neglected and understudied disease. The causative agent of Leptospirosis was first identified in 1916 but the slow in vitro growth rate and limited genetic tools with which to manipulate the genome of this spirochete have hampered the identification of virulence factors and development of a vaccine. Leptospires can be divided into three subgroups: saprophytes, pathogens, and a middle group of unknown pathogenicity. The most widely used and studied species are L. biflexa (a free-living, non-pathogenic saprophyte) and L. interrogans (a pathogen). However, the non-pathogenic L. biflexa is more easily cultivated and more amenable to genetic manipulation than the pathogenic L. interrogans. Therefore, we have initially focused on L. biflexa to master the microbial and genetic techniques needed to manipulate this genus, with the intention to transfer this expertise to the more refractory pathogenic strains. Targeted gene inactivation, shuttle vector transformation, and transposon mutagenesis have all been successfully used in L. biflexa. To date, no shuttle vector system exists for pathogenic species and there are only two published reports of targeted gene inactivation in L. interrogans. Transposon mutagenesis can be applied to L. interrogans but it functions at such a low efficiency that it cannot be utilized for any broad applications, such as auxotrophic screens or signature tagged mutagenesis. The lack of a shuttle vector for L. interrogans hinders complementation and thus limits interpretation of any resulting phenotypes of transposon or targeted deletion mutants. Our focus has therefore concentrated on increasing and improving the molecular genetic tools available to manipulate leptospires, relying on our experience in developing a genetic system for another spirochete, Borrelia burgdorferi, the causative agent of Lyme disease. The long-term objective of this project is to use the improved tools and techniques to understand the mechanisms of infection and pathogenecity of L. interrogans and accelerate the development of preventative measures against Leptospirosis. Our current work in L. biflexa is now focused on more fully developing it as a model organism for the pathogenic leptospires. Since L. biflexa has a better transformation frequency than other species we plan to optimize new techniques in this organism. However, as a model organism, key information is lacking in this system, specifically regarding what proteins are physiologically important or highly expressed during in vitro cultivation. Therefore, our approach has been to first optimize culture conditions and transformation techniques, followed by proteome mapping and directed mutagenesis against specific targets of interest. In FY2011, Mr. Hunter Stone and Dr. Amit Sarkar optimized the growth conditions for culturing and selecting for Leptospira spp. This included adapting a recipe for EMJH medium that allows Leptospira growth when made from basic ingredients. Previous attempts to make our own medium had failed but by testing various components, we identified the Fe++ and antibiotic concentrations as too high to support sustained Leptospira growth. By testing various concentrations of different media components, we were able to optimize the recipe. We obtained a variety of saprophytic and pathogenic strains from different labs and found they were able to grow in both commercially purchased medium and in our in-house medium. Finally, Hunter modified an existing shuttle vector to simplify genetic manipulations by adding a multiple cloning site region and the -galactosidase gene for color screens. With culture conditions optimized and the construction of a more amenable cloning vector, we proceeded in 2012 to use the saprophyte L. biflexa to perform genetic transformation procedures including shuttle vector transformation and targeted gene inactivation. Using allelic exchange techniques, we engineered deletion mutants in the batABD locus, genes that encode proteins proposed to play a role in protecting some bacteria from oxidative stress. We compared the wild-type strain and deletion mutants under various oxidative stress conditions and found that the data do not support a protective role for the Leptospira Bat proteins in directly coping with oxidative stress, as previously proposed. Further, we demonstrated that L. biflexa is relatively sensitive to reactive oxygen species such as H2O2, suggesting that this spirochete lacks a strong, protective defense against oxidative damage despite being a strict aerobe. These results are described in a manuscript submitted for publication. Currently, we are developing a global proteomic map of in vitro cultivated L. biflexa to identify highly expressed proteins from membrane and soluble cellular fractions. Highly expressed proteins allow us to identify targets that may play important physiological roles and also use as tagged proteins for various expression studies. We have also begun to experiment directly in the pathogenic strain L. interrogans, studying homologs that have been shown in other organisms to target and degrade foreign DNA. This system may help explain why transformation frequencies are much lower in pathogenic strains where this system is present, versus free-living strains that lack these homologs. Now having mastered the techniques to cultivate and manipulate Leptospira spp. we have completed one project studying the oxidative stress response of the model organism L. biflexa and continued to develop our basic knowledge of this organism by mapping its proteome. Carrying these techniques and knowledge on to the pathogenic strains should help to expand the genetic tools needed for elucidating mechanisms of infection and pathogenicity.